Modified mite allergen and pharmaceutical uses thereof

ABSTRACT

The present invention provides a modified  Dermatophagoides pteronyssinus  allergen Der p 5 protein which has ability to inhibit IgE binding when exposed against to the antigen. A method for treating allergy comprising administrating a therapeutically effective dose of the modified  D. pteronyssinus  allergen Der p 5 protein to a subject suffering from allergy Der p 5 is also provided.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention mainly relates to a modified allergen obtained by alteringDer p 5 allergen of house dust mite and the pharmaceutical uses thereof.

2. Description of the Related Art

Allergy refers to an acquired potential to develop immunologicallymediated adverse reaction to normally innocuous substances. Allergicreaction provokes symptoms such as itching, coughing, wheezing,sneezing, watery eyes, inflammation and fatigue. Many allergic diseasesare due to several kinds of symptoms which are developed bysensitization to the antigen causing the diseases. In an allergicdisease, an IgE antibody specific for an allergen (e.g. pollens and mitedust) in blood serum and tissue is produced, and when the antibody isexposed again to the antigen, the antibody reacts with the antigen ineach tissue. It is normally believed that an allergic reaction includesan early specific immune response and a late inflammatory reaction. Itis reported that an allergen mediates the early phase of allergy bystimulating high affinity immunoglobulin (IgE) receptors. Mast cells andbasophils, when stimulated by allergens, will release histamine andcytokines. The cytokines released from mast cells and basophils thenmediate the late phase of allergy by recruiting inflammatory cells.

It is reported that allergic diseases, such as bronchial asthma,childhood asthma, atopic dermatitis and the like, are mainly caused byallergens from mites living in house dust. Several kinds of proteins ofmite allergens, such as Der p 1 and Der p 2, have been identified. Der p5 is a 14-k Da group 5 mite allergen which contains a 19-residue leaderprotein and a 113-residue mature protein was cloned and sequenced (Linet al., Allergens, IgE, mediators, inflammatory mechanisms. J AllergyClin Immunol 1994; 6:989-996). Although only 60% of mite-allergicchildren reacted to Der p 5, the IgE reactivity appeared to be strongerthan that of Der p 1 and Der p 2 in Taiwan. Furthermore, among thevarious allergic diseases, the group of children with asthma havesignificant more reactivity than the group with rhinitis alone. Der p 5is regarded as a clinically significant allergen in mite allergy.

Various therapies have been pursued in order to treat the symptoms ofallergies. Particularly, “oral tolerance” is considered to be an idealcandidate for treatment of an allergic disease. Oral tolerance has beencharacterized as a state of antigen-specific systemic immunologicalunresponsiveness or tolerance, which is induced by prior oraladministration or feeding of antigen. The primary mechanism by which anorally administered antigen induces tolerance is believed to be via thegeneration of active suppression or clonal anergy.

However, directly administrating wild-type mite allergens may raise anallergic reaction, namely anaphylactic shock, in hyposensitizationtherapy, because the activity of these wild-type allergens is high. Ifthe binding between the antigen and the IgE antibody is controlled, thecross-linking among the IgE antibodies on mast cells or basophils, andthe release of histamine and cytokines are controlled to treat allergicdiseases. Regarding Der p 1 and Der p 2, the B-cell epitopes and T-cellepitopes have been demonstrated, and the side-directed mutagenesis ofDer p 1 and Der p 2 in order to inhibit IgE binding when exposed againto the antigen has been disclosed in U.S. Pat. No. 6,187,311, butnothing on Der p 5 has been disclosed.

SUMMARY OF THE INVENTION

The invention provides a modified Dermatophagoides pteronyssinusallergen Der p 5 protein, which provides an efficacy in inhibiting IgEbinding when exposed again to the antigen in a subject or has a lowerallergen activity than wide-type allergens. Therefore, the inventionprovides a method for treating an allergic disease using the modified D.pteronyssinus allergen Der p 5 protein, in which no anaphylactic shockshows.

One subject of the invention is to provide a modified Dermatophagoidespteronyssinus allergen Der p 5 protein comprising some substitutions atone or more residue positions corresponding to the 44^(th), 77^(th),99^(th) and 103^(rd) amino acid residues of the wild-type D.pteronyssinus allergen Der p 5 protein, in order to minimize thestructural change and provide an efficacy in inhibiting IgE binding whenexposed again to the antigen in a subject and reducing in vivo allergicreactivity but retaining the ability to trigger peripheral bloodmononuclear cell (PBMC) proliferation. The modified mite allergen Der p5 protein according to the invention can inhibit IgE binding whenexposed again to the antigen.

Another object of the invention is to provide an isolated nucleic acidmolecule having a nucleotide sequence encoding the modifiedpteronyssinus allergen Der p 5 protein according to the invention. Inone embodiment of the invention, the isolated DNA molecule comprises oneor more substitutions of the nucleotide sequence of SEQ ID NO:1.

In another aspect, the invention provides a host cell comprising theisolated nucleic acid molecule.

The present invention involves a pharmaceutical composition for treatingallergic diseases in association with Der p 5 or immunizing againstallergic diseases in association with Der p 5, comprising atherapeutically effective dose of the modified D. pteronyssinus allergenDer p 5 protein according to the invention and a pharmaceuticallyacceptable carrier.

The present invention also involves a method for treating allergicdiseases in association with Der p 5 which comprises administrating atherapeutically effective dose of the modified D. pteronyssinus allergenDer p 5 protein according to the invention to a subject suffering fromallergic diseases, particularly in association with Der p 5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the result of the wild-type Der p 5 antigenicitysimulation.

FIG. 2 illustrates the result of the wild-type Der p 5 with severalsubstitutions, in which the antigenicity simulation was changed.

FIG. 3 illustrates the electrophoresis results of the wild type andmodified Der p 5 resolved on an SDS-polyacrylamide gel.

FIG. 4 illustrates the result of IgE inhibition ELISA, wherein Apresents 10 μg Der p 5; B presents 50 μg Der p 5; C presents 10 μgDp5p-m2; D presents 50 μg Dp5p-m2; E presents 10 μg Dp5p-m3; F presents50 μg Dp5p-m3; G presents 10 μg Dp5p-m4; and H presents 50 μg Dp5p-m4.

FIG. 5 illustrates the result of intradermal skin test.

FIGS. 6 a and 6 b illustrate the result of PBMC proliferation assay ofPatient 1 and Patient 2, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The aim of the invention is to produce a hypoallergenic form of Der p 5.

The invention provides a modified Dermatophagoides pteronyssinusallergen Der p 5 protein, which provides an efficacy in inhibiting IgEbinding when exposed again to the antigen in a subject or has a lowerallergen activity than wide-type allergens. In one embodiment of theinvention, the wild-type allergen has the nucleotide sequence of SEQ IDNO:2. According to the invention, the substitutions are at one or moreresidue positions corresponding to the 44^(th), 77^(th), 99^(th), and103^(rd) amino acid residues of the wild-type mite allergen Der p 5protein.

The term “modified Dermatophagoides pteronyssinus allergen” used hereinrefers to a gene-engineered mite allergen of which the amino acidsequence is different from that of wild-type allergen. The modifiedallergen of the present invention can be produced by any method suitableto the aims of the present invention, and preferably by a site-directedmutagenesis method, and more preferably a PCR method. For example, the44^(th) proline residue of the modified Der p 5 protein (SEQ ID NO:4) isreplaced with an alanine residue using the DNA chain as shown in SEQ IDNO:3.

In a preferred embodiment of the invention, sites to be modified of themite allergen (Der p 5) are estimated both in antigenicity andhydrophilicity. The term “antigenicity” used herein refers to ability ora tendency to elicit an allergic response. The antigenicity usuallyrelates to IgE binding ability, B cell binding ability, T cell bindingability, and peripheral blood mononuclear cell (PBMC) proliferationstimulating ability. Epitopes of antigen play a crucial role inantigenicity, and the three-dimensional configuration and hydrophilicityare also important. It is acceptable to decrease the antigenicity bychanging the configuration of the epitopes but retaining the samehydrophilicity and structure of the total allergen. Many commercialcomputer software packages are well established for simulating thetheoretical antigenicity of an antigen such as MacVector 6.05 computermodel.

Several amino acid residues of Der p 5 are shown to dominate theantigenicity, which comprise the aspartic acid at the 2^(nd) position,the aspartic acid at the 6^(th) position, the glutamine at the 8^(th)position, the proline at the 44^(th) position, the lysine at the 77^(th)position, the arginine at the 99^(th) position, and the lysine at the103^(rd) position, wherein preferably the sites are selected from thegroup consisting of the 44^(th), 77^(th), 99^(th), and 103^(rd)positions. According to the invention, the modified and hypoallergenicforms of Der p 5 are obtained by changing at least one the amino acidresidue at these positions in the wild-type mite allergen Der p 5. In amore preferred embodiment, the modified mite allergen Der p 5 has achanged site at the 44^(th) position that is proline in the wild type.

Amino acid residues selected to replace the residues have differentfunctional group in side chain from those in the wild-type Der p 5. Inone embodiment of the invention, at least one of the residue of the wildtype is substituted by an amino acid with an alkyl side chain, and in apreferred embodiment of the invention, the residue is substituted byalanine.

According to the invention, an isolated nucleic acid molecule having anucleotide sequence encoding the modified Dermatophagoides pteronyssinusallergen Der p 5 protein according to the invention is also provided formanipulations, expression and storage. The isolated DNA molecule can beexpressed by using a suitable vector, which can be expressed in a hostcell. The term “host cell” used herein refers to a prokaryotic oreukaryotic cell which is transformed or transfected with the vectorcomprising the desired genes, wherein preferably, the host cell canexpress the desired genes to producing the gene products.

A method of producing the modified Dermatophagoides pteronyssinusallergen Der p 5 protein according to the invention is provided, whichcomprises culturing the host cell carrying the isolated nucleic acidmolecule according to the invention under conditions such that saidmodified Dermatophagoides pteronyssinus allergen Der p 5 protein isexpressed and produced thereby, and isolating said modifiedDermatophagoides pteronyssinus allergen Der p 5 protein.

According to the invention, the modified Dermatophagoides pteronyssinusallergen Der p 5 protein changed in antigenicity can be used in treatingDer p 5 allergic disease which comprises administrating atherapeutically effective dose of the modified Dermatophagoidespteronyssinus allergen Der p 5 to a subject suffering from Der p 5allergic disease. Because the antigenicity of the modified mite allergenis reduced, anaphylactic shock which may be raised in directlyadministrating wild-type mite allergens in hyposensitization therapy isavoided. The binding between the antigen and the IgE antibody iscontrolled and the cross-linking among the IgE antibodies on mast cellsor basophils, and the release of chemical mediators are controlled totreat allergic diseases. However, the ability to stimulate PBMCproliferation is still retained.

The term “allergic disease” used herein refers to allergy in associationwith Der p 5. The allergic disease includes rhinitis, sinusitis, asthma,hypersensitive pneumonia, extrinsic allergic alveolitis, conjunctivitis,urticaria, eczema, dermatitis, anaphylaxis, angioedema, allergic andmigraine headache, and certain gastrointestinal disorders.

The following Examples are given for the purpose of illustration onlyand are not intended to limit the scope of the present invention.

EXAMPLE 1 Modified Mite Allergen Der p 5 Construction

Antigenicity of Der p 5 simulation: The theoretical IgE epitopes of Derp 5 were predicted with MacVector 6.05 computer model. The amino acidsequence of wild-type Der p 5 was input and calculated according toAntigenic Index listed below (according to MacVector operating guide):Antigenic Index=Σ{0.3 hydro[i]+0.15surf_prob[i]+0.15 flex[i]+0.2chou_fas[i]+0.2rob_garn[i]}

The antigenicity of wild-type Der p 5 simulated was shown in FIG. 1.Several sites regarded as having high antigenicity comprising D², D⁶,Q⁸, P⁴⁴, K⁷⁷, R⁹⁹, and K¹⁰³ were replaced with alanine residue and theninput the new amino acid sequence for simulation. The result was shownin FIG. 2. According to the simulation result, the Der p 5 with thesesites changed had low antigenicity. For the reason, mutations at thesesites were candidates for constructing the hypoallergenic form of Der p5.

Site-directed mutagenesis: Wild-type Der p 5 gene (SEQ ID NO:1) wascloned in a pQE 60 vector and then mutated by using the QuickChangeSite-Directed Mutagenesis Kit (Stratagene). The method for constructingthe modified Der p 5 was described in the instruction manual of the kit.Dp5p-m2 (Der p 5 with the proline residue at position 44 replaced withalanine) were constructed by using DP5-2F5′-CATTTTGAAGAAAAAGCGACAAAAGAAATGAAAG-3′ (SEQ ID NO:9) and DP5-2R5′-CTTTCATTTCTTTTGTCGCTTTTTCTTCAAAATG -3′ (SEQ ID NO:10). The codon CCGwas changed to GCG and the proline was changed to alanine. Dp5p-m3 (Derp 5 with the lysine residue at position 77 replaced with alanine) wereconstructed by using DP5-3F 5′-GATCGTCTTATGCAACGTGCAGATTTAGATA-3′ (SEQID NO:11) and DP5-3R 5′-TATCTAAATCTGCACGTTGCATAAGACGAT-3′ (SEQ IDNO:12). The codon AAA was changed to GCA and the lysine was changed toalanine. Dp5p-m4 (Der p 5 with the arginine residue at position 99 andthe lysine residue at position 103 replaced with alanine) wereconstructed by using DP5-4F5′-CATCACGGATCCGAAGCTAAAAAACATGATTATGCGAAT-3′ (SEQ ID NO:13) and DP5-4R5′-CGCGCAAGCTTTTAAACTTCAATCTTTTTAACACGTGCTTCTTCT GCTTTCAAATCAGCTTC-3′(SEQ ID NO:14). The codon usage CGT is changed to GCT and AAA is changedto GCA, and thus the arginine and lysine are both changed to alanine.

Protein expression: The plasmids containing Der p 5, Dp5p-m2, Dp5p-m3,and Dp5p-m4 were transformed into Escherichia coli (M15) XL1-Blue forexpression. The bacteria were cultured and the proteins expressed weresubjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The protein sizes of the modified allergen and that of thewild-type Der p 5 were the same (about 14 k Da). The result show thatthe side-directed mutagenesis was successfully performed (referring toFIG. 3).

EXAMPLE 2 IgE Binding Inhibition of the Modified Mite Allergens

The method for assaying IgE binding inhibition of the modified miteallergens was based on the description by Beezhold et al. (Beezhold etal., Mutational analysis of the IgE epitopes in the latex allergen Hev b5. J Allergy Clin Immunol 2001; 107:1069-1076). Sera from six miteallergy patients who were highly reactive to Der p 5 were taken for theassay. Standardized wild type mite allergen Der p 5 were coated on anELISA plate, and then blocked. The sera were mixed with the wild-typeand modified mite allergens (as inhibitors) and then reacted with Der p5 coated on the ELISA plate. Biotin-labeled anti-human IgE was thenadded. Streptavidin-Alkaline phosphatase (Streptavidin-AKP) was labeledand p-Nitrophenyl phosphate (pNpp) was then added as substrate. Theabsorbance at 405 nm was measured. The percentage inhibition wascalculated as below:[1−(OD of Inhibitor−OD of Background)/(OD of Non-Inhibition−OD ofBackground)]×100%

The result of IgE inhibition ELISA was shown in FIG. 4. Comparing thewild type and the modified allergens, Dp5p-m2 had stronger ability toinhibit IgE binding when exposed again to the antigen.

EXAMPLE 3 Intradermal Skin Test

The method for assaying intradermal skin test was similar with thedescription by van Hage-Hamsten et al. (van Hage-Hamsten et al., Skintest evaluation of genetically engineered hypoallergenic derivatives ofthe major birch pollen allergen, Bet v 1: Results obtained with a mix oftwo recombinant Bet v 1 fragments and recombinant Bet v 1 trimer in aSwedish population before the birch pollen season. J Allergy ClinImmunol 1999; 104:969-977). Eight Der p 5 sensitive mite allergypatients were chosen. The wild type and modified Der p 5 were diluted to1 mg/mL, and 0.05 mL of allergens was taken for each injection site onthe patients' forearms 5 cm apart. A reaction of greater than 5×5 mm(diameter) for 15 min. after injection was regarded as a positiveresponse.

The result of intradermal skin test was shown in FIG. 5. All of themodified Der p 5 had lower allergic response than the wild type in theintradermal skin test.

EXAMPLE 4 PBMC Proliferation Assay

The PBMC proliferation assay was performed by using Cell ProliferationELISA, BrdU (calorimetric) kit (Roche), and the method was described inthe instruction manual of the kit. PBMCs at various concentrations (fromDer p 5 sensitive mite allergy Patient 1 and Patient 2) were culturedwith wild-type and modified Der p 5 for 2 days. PBMCs cultured withPhaseolus vulgaris agglutinin (PHA) were as the positive control andPBMCs cultured only were as the negative control. The cells were pulsedwith BrdU and harvested overnight, and then anti-BrdU-peroxidase (POD)was added. Tetramethyl-benzidine (TMB) as substrate was added and thespectrum of mixture was measure at 655 nm.

The results of the PBMC proliferation assay were shown in FIG. 6 a(Patient 1) and FIG. 6 b (Patient 2). The numbers of PBMC cultured withwild-type and modified Der p 5 were similar. It was shown that thestimulating PBMC proliferation ability of the modified Der p 5 wasretained.

While embodiments of the present invention have been illustrated anddescribed, various modifications and improvements can be made by personsskilled in the art. It is intended that the present invention is notlimited to the particular forms as illustrated, and that all themodifications not departing from the spirit and scope of the presentinvention are within the scope as defined in the appended claims.

1. An isolated nucleic acid molecule having a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 3, 5 and
 7. 2. A hostcell comprising the isolated nucleic acid molecule according to claim 1.